Two-step transplantation with adipose tissue-derived stem cells increases follicle survival by enhancing vascularization in xenografted frozen-thawed human ovarian tissue

Diego Daniel Manavella (BE), Luciana Cacciottola (IT), Christiani Amorim (BE), Jacques Donnez (BE), Marie-Madeleine Dolmans (BE)

[Manavella] Pôle de Recherche en Gynécologie, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain, [Cacciottola] Università degli Studi di Milano, [Amorim] Pôle de Recherche en Gynécologie, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain, [Donnez] Society for Research into Infertility, [Dolmans] Pôle de Recherche en Gynécologie, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain

Context Ovarian tissue transplantation for fertility preservation has been gaining popularity over the past decade thanks to increasingly successful outcomes. However, follicle loss in the early post-grafting period limits the life span of grafts. Improving the grafting procedure has been the main focus of research aiming to increase follicle survival rates. Objective To evaluate the short-term effects of a novel two-step ovarian tissue transplantation approach using adipose tissue-derived stem cells (ASCs) to enhance graft vascularization and ultimately increase follicle survival rates. Methods A total of 15 severe combined immunodeficient mice were intraperitoneally grafted with frozen-thawed human ovarian tissue. Levels of partial pressure of oxygen (pO2) were monitored in vivo by electron paramagnetic resonance (EPR) oximetry on post-grafting days 3 (n=15) and 7 (n=15). Samples for histology and immunohistochemistry were collected after euthanizing the mice on day 7. One piece of ovarian tissue per patient served as non-grafted controls. Patients Ovarian tissue from 5 patients (aged <35). Interventions A peritoneal grafting site had been previously prepared in a first step using either empty fibrin (Fi+OT group [n=5]) or ASC-loaded fibrin (Fi/ASCs+OT group [n=5]) for 14 days prior to ovarian tissue grafting. Five mice were grafted using the standard one-step transplantation procedure and served as controls (OT group). Main outcome measures Graft pO2 levels measured by EPR oximetry on post-grafting days 3 and 7. On day 7: follicle density (hematoxylin and eosin); graft vascularization (double anti-human/mouse CD34); follicle growth (anti-human Ki67); follicle apoptosis (TUNEL assay); and fibrosis (Masson’s trichrome). Results Significantly higher pO2 levels were observed in the Fi/ASCs+OT group compared to the OT group (p=0.028). Total CD34-positive vessel area was significantly greater in the Fi/ASCs+OT group (p < 0.05). Significantly increased follicle survival rates and significantly fewer apoptotic follicles were found in the Fi/ASCs+OT group (p < 0.05). Significantly higher numbers of Ki67-positive primordial follicles were encountered in all grafted groups (p < 0.01). No difference was observed in fibrosis. Conclusion Our two-step transplantation approach using ASCs evidenced higher rates of oxygenation and vascularization in grafted ovarian tissue in the early post-grafting period, leading to decreased apoptosis and increased follicle survival rates.